Nonintegrative sustained expression

Robust system for mcDNA

The Minicircle advantage

Minicircles are episomal DNA vectors that are produced as circular expression cassettes devoid of any bacterial plasmid DNA backbone. Their smaller molecular size enables more efficient transfections and offers sustained expression over a period of weeks as compared to standard plasmid vectors that only work for a few days.

The Minicircle DNA elements are generated by an intramolecular (cis-) recombination from a parental plasmid (PP) mediated by PhiC31 integrase. The full-size MC-DNA construct is grown in a special host E. coli bacterial strain ZYCY10P3S2T (catalog#MN900A-1). This highly engineered strain harbors an Arabinose-inducible system to express the PhiC31 integrase and the I-SceI endonuclease simultaneously. The ZYCY10P3S2T strain also contains a robust arabinose transporter LacY A177C gene. Adding arabinose to the media turns on the integrase and endonuclease genes. The PhiC31 integrase produces the MC-DNA molecules as well as PP-DNA from the full-size MC-DNA construct. The Sce-I endonuclease then degrades the PP-DNA backbone.

The Parental Plasmid DNA (PP) contains several engineered I-SceI restriction sites that ultimately lead to the destruction of the PP-DNA but not the MC-DNA. The difference between MC and standard plasmid vectors is that the MC no longer contains the bacterial origin of replication or the antibiotic resistance markers. Sequences within the bacterial plasmid backbone contain signals for methylation and transgene silencing. Thus delivering only the minicircles to cells lengthens the expression of the transgene over traditional transient transfections of plasmids.

For dividing cells, expression of the minicircles lasts up to 14 days. For non-dividing cells, expression of the minicircles drops slightly after the first week, but then can continue expressing the transgenes for months.